Antibiotic compounds

ABSTRACT

This invention relates to novel purified compounds of Formula I. The invention includes all stereoisomeric forms and all tautomeric forms of the compounds of Formula I and pharmaceutically acceptable salts and derivatives. The present invention further relates to processes for the production of the novel antibacterial compounds by fermentation of the microorganism belonging to  Streptomyces  species (PM0626271/MTCC 5447) and to pharmaceutical compositions containing one or more of the novel compounds as active ingredient and their use in medicines for treatment and prevention of diseases caused by bacterial infections.

CROSS REFERENCE APPLICATIONS

This is a divisional of copending application Ser. No. 13/393,294 filedon 29 Feb. 2012, which is a 371 of International ApplicationPCT/IB2010/053897 filed on 31 Aug. 2010, which designated the U.S.,claims the benefit thereof and incorporates the same by reference. Thenonprovisional application designated above, namely application Ser. No.13/393,294 filed 29 Feb. 2012, claims the benefit of U.S. ProvisionalApplication No. 61/239,186 filed 9 Sep. 2009 and incorporates the sameby reference.

FIELD OF THE INVENTION

This invention relates to novel compounds of Formula I havingantibacterial activity. The compounds may be obtained by fermentation ofa microorganism belonging to Streptomyces species (PM0626271/MTCC 5447).The invention also includes all stereoisomeric forms and all tautomericforms of compounds of Formula I and pharmaceutically acceptable saltsand derivatives thereof. The present invention further relates toprocesses for the production of the novel antibacterial compounds and topharmaceutical compositions containing one or more of the novelcompounds as an active ingredient and their use in medicines fortreatment and prevention of diseases caused by bacterial infections.

BACKGROUND OF THE INVENTION

The dramatic rise in the prevalence of antibiotic resistance amongbacteria currently poses a serious threat to public health worldwide. Ofparticular concern are infections caused by methicillin-resistantStaphylococcus aureus (MRSA), penicillin-resistant Streptococcuspneumoniae (PRSP), vancomycin-resistant Enterococcus (VRE) (Clin.Microbiol. Infect., 2005, 11, Supplement 3: 22-28) and multi drugresistant (MDR) Mycobacterium tuberculosis (Eur. Respir. J., 2002,Supplement 36, 66S-77S).

Thiostrepton, an antibiotic isolated from Streptomyces azureus, has beenreported to be an effective anti-infective medicine having the samegeneral antibiotic spectrum as penicillin and is used againstgram-positive coccal infections (U.S. Pat. No. 2,982,689).

Siomycin, a sulfur-containing peptide antibiotic isolated fromStreptomyces sioyaensis, has been reported to be active againstgram-positive bacteria and mycobacteria with little or no activityagainst gram-negative bacteria (The Journal Of Antibiotics, 1969,364-368).

There is a need to discover new compounds, which can be used as drugs totreat patients who are at risk of infection or are infected withbacteria, especially multi drug resistant bacteria such as MRSA, VRE andMycobacterium tuberculosis.

SUMMARY OF THE INVENTION

The present invention relates to novel compounds of Formula I.

The present invention also relates to novel purified compounds ofFormula I, isolated from the fermented broth of the microorganismbelonging to Streptomyces species (PM0626271/MTCC 5447).

The invention also relates to all stereoisomeric forms and alltautomeric forms of compounds of Formula I and pharmaceuticallyacceptable salts and derivatives thereof.

The compounds of Formula I, and isomers, pharmaceutically acceptablesalts and derivatives thereof, have antibacterial activity and areuseful for the treatment or prevention of diseases caused by bacteria,particularly multi drug resistant bacteria such as MRSA, VRE andMycobacterium tuberculosis.

The invention further relates to pharmaceutical compositions comprisingone or more of the novel compounds of Formula I, an isomer, apharmaceutically acceptable salt, or derivative thereof, as an activeingredient for the treatment of diseases caused by bacteria,particularly multi drug resistant bacteria such as MRSA, VRE andMycobacterium tuberculosis.

The present invention further relates to processes for the production ofthe compounds of Formula I and/or their isomers from the microorganismbelonging to Streptomyces species (PM0626271/MTCC 5447).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Illustrates the ¹H NMR spectrum (500 MHz; Instrument Bruker) ofcompound of Formula I(a) in CDCl₃:CD₃OD (4:1).

FIG. 2 Illustrates the ¹³C NMR spectrum (75 MHz; Instrument Bruker) ofcompound of Formula I(a) in CDCl₃:CD₃OD (4:1).

FIG. 3 Illustrates the ¹H NMR spectrum (500 MHz; Instrument Bruker) ofcompound of Formula I(b) in CDCl₃:CD₃OD (4:1).

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it has to beunderstood that this invention is not limited to particular embodiments.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly indicatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of the ordinaryskill in the art to which the invention belongs.

As used herein, the term “derivative” refers to a compound that isderived from a similar compound or a compound that can be imagined toarise from another compound, if one atom is replaced with another atomor group of atoms.

As used herein, the term “stereoisomer” refers to all isomers ofindividual compounds that differ only in the orientation of their atomsin space. The term stereoisomer includes mirror image isomers(enantiomers), mixtures of mirror image isomers (racemates, racemicmixtures), geometric (cis/trans or syn/anti or E/Z) isomers, and isomersof compounds with more than one chiral center that are not mirror imagesof one another (diastereoisomers). The compounds of the presentinvention may have asymmetric centers and occur as racemates, racemicmixtures, individual diastereoisomers, or enantiomers, or may exist asgeometric isomers, with all isomeric forms of said compounds beingincluded in the present invention.

As used herein, the term “tautomer” refers to the coexistence of two (ormore) compounds that differ from each other only in the position of one(or more) mobile atoms and in electron distribution, for example,keto-enol and imine-enamine tautomers.

As used herein, the term “fermented broth” refers to a suspension ofmicrobial culture in a nutrient medium containing compounds produced bythe microbes during its growth and also having unconsumed nutrients.

As used herein, the term “mutant” refers to an organism or cell carryinga mutation, which is an alternative phenotype to the wild-type.

As used herein, the term “variant” refers to an individual organism thatis recognizably different from an arbitrary standard type in thatspecies.

The novel compounds of Formula I are structurally represented by thefollowing formula:

The novel compound of Formula I(a) has the molecular formulaC₇₁H₈₃N₁₉O₁₈S₅ (molecular weight 1649.5). The novel compound of FormulaI(b) has the molecular formula C₇₁H₅N₁₉O₁₈S₅ (molecular weight 1651.5).The novel compounds of Formula I(a) and Formula I(b) may becharacterized by any one or more of their physico-chemical and spectralproperties, such as high performance liquid chromatography (HPLC), massspectrum (MS), infra red (IR) and nuclear magnetic resonance (NMR)spectroscopic data as discussed herein below.

The structure of the novel compounds of Formula I(a) and Formula I(b)has been elucidated and its complete characterization is done by HPLC,high resolution MS (HRMS), IR and NMR spectroscopic data. The compoundsof Formula I(a) and Formula I(b) are new antibiotics active againstbacteria, particularly multi drug resistant bacteria such as MRSA, VREand Mycobacterium tuberculosis. Compounds of Formula I(a) and FormulaI(b) have hitherto unreported structures.

The microorganism, which may be used for the production of the compounds

of Formula I(a) and Formula I(b), is a strain of Streptomyces species(PM0626271/MTCC 5447), herein after referred to as culture no.PM0626271, isolated from a soil sample collected from Schirmacher Oasisin Antarctic region.

The present invention further provides processes for the production ofthe compounds of Formula I(a) and Formula I(b) from culture no.PM0626271, comprising the steps of: cultivating the culture no.PM0626271 under submerged aerobic conditions in a nutrient mediumcontaining one or more sources of carbon and one or more sources ofnitrogen and optionally nutrient inorganic salts and/or trace elements;isolating the compounds of Formula I(a) and Formula I(b) from thefermented broth; and purifying the compounds of Formula I(a) and FormulaI(b) using purification procedures generally used in the related art.

Preliminary identification of culture no. PM0626271, which is theproducer of compounds of Formula I(a) and Formula I(b) was performed byexamination of its colony morphology, wet mount observations and Gramstain reaction. Microscopic studies on the strain of isolated culture noPM0626271 were carried out on Actinomycete Isolation agar (AS-AIA;details given in Example section) containing 1.5% agar and observationswere made at 1, 2 and 3 days of incubation at 25° C.

Growth on AS-AIA containing 1.5% agar develops as 1 mm diameter colonieswith white sporulation, scanty yellowish substrate mycelia, slightlyraised appearance, no diffusible pigment and back side dark buff incolor. Under phase contrast light microscopy, wavy tangles of thinmycelia with sporulated tips are observed at 400× magnification. Theyare Gram-positive. The free spores are non-motile. The observedmorphology classifies this organism as a member of Streptomycetesfamily.

Culture no. PM0626271 has been deposited with Microbial Type CultureCollection (MTCC), Institute of Microbial Technology, Sector 39-A,Chandigarh—160 036, India, a World Intellectual Property Organization(WIPO) recognized International Depository Authority (IDA) and has beengiven the accession number MTCC 5447.

In addition to the specific microorganism described herein, it should beunderstood that mutants, such as those produced by the use of chemicalor physical mutagens including X-rays, U.V. rays etc. and organismswhose genetic makeup has been modified by molecular biology techniques,may also be cultivated to produce the compounds of Formula I(a) andFormula I(b).

The screening for suitable mutants and variants which can produce thecompound according to the invention can be confirmed by HPLC and/ordetermination of biological activity of the active compounds accumulatedin the fermented broth, for example by testing the compounds forantibacterial activity.

The medium and/or nutrient medium used for isolation and cultivation ofculture no. PM0626271, which produces the compounds of Formula I(a) andFormula I(b), preferably contains sources of carbon, nitrogen andnutrient inorganic salts. The carbon sources are, for example, one ormore of starch, glucose, sucrose, dextrin, fructose, molasses, glycerol,lactose, or galactose. Preferred carbon sources are soluble starch andglucose. The sources of nitrogen are, for example, one or more ofsoybean meal, peanut meal, yeast extract, beef extract, peptone, maltextract, corn steep liquor, gelatin, or casamino acids. Preferrednitrogen sources are peptone and yeast extract. The nutrient inorganicsalts are, for example, one or more of sodium chloride, potassiumchloride, calcium chloride, magnesium chloride, ferric chloride,strontium chloride, cobalt chloride, potassium bromide, sodium fluoride,sodium hydrogen phosphate, potassium hydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, calcium carbonate, sodiumbicarbonate, sodium silicate, ammonium nitrate, potassium nitrate,ferrous sulphate, sodium sulphate, ammonium sulphate, magnesiumsulphate, ferric citrate, boric acid or trace salt solution. Calciumcarbonate, sodium chloride, and magnesium chloride are preferred.

The maintenance of culture no. PM0626271 may be carried out at atemperature ranging from 22° C. to 36° C. and a pH of about 7.5 to 8.0.Typically, culture no. PM0626271 is maintained at 25° C. to 27° C. and apH of about 7.4 to 7.8. The well-grown cultures may be preserved in therefrigerator at 4° C. to 8° C.

Seed culture cultivation of culture no. PM0626271 may be carried out ata temperature ranging from 25° C. to 36° C. and a pH of about 7.5 to 8.0for 66 hours to 75 hours at 200 rpm (revolutions per minute) to 280 rpm.Typically, culture no. PM0626271 seed is cultivated at 29° C. to 31° C.and a pH of about 7.4 to 7.8, for 72 hours at 230 rpm to 250 rpm.

The production of the compounds of Formula I(a) and Formula I(b) may becarried out by cultivating culture no PM0626271 by fermentation at atemperature ranging from 26° C. to 36° C. and a pH of about 6.5 to 8.5,for 24 hours to 96 hours at 60 rpm to 140 rpm and 100 lpm (liter perminute) to 200 lpm aeration. Typically, culture no. PM0626271 iscultivated at 30° C. to 32° C. and pH 7.4 to 7.8 for 40 hours to 96hours at 90 rpm and 110 lpm aeration.

The production of the compounds of Formula I(a) and Formula I(b) can becarried out by cultivating culture no. PM0626271 in a suitable nutrientbroth under conditions described herein, preferably under submergedaerobic conditions, for example in shake flasks, as well as inlaboratory fermenters. The progress of fermentation and production ofthe compounds of Formula I(a) and Formula I(b) can be detected by highperformance liquid chromatography (HPLC) and by measuring thebioactivity of the fermented broth against Staphylococci and/orEnterococci species by the known microbial agar plate diffusion assaymethod. The preferred culture is Staphylococcus aureus E710, which is astrain resistant to methicillin, a β-lactam antibiotic reported in theliterature, and Enterococcus faecium R2 (VRE) which is resistant tovancomycin. In the resulting fermented broth, the compounds of FormulaI(a) and Formula I(b) are present in the culture filtrate as well as incell mass and can be isolated using known separation techniques such assolvent extraction and column chromatography. Thus, the compounds ofFormula I(a) and Formula I(b) can be recovered from the culture filtrateby extraction at a pH of about 5 to 9 with a water immiscible solventsuch as petroleum ether, dichloromethane, chloroform, ethyl acetate,diethyl ether or butanol, or by hydrophobic interaction chromatographyusing polymeric resins such as “Diaion HP-20®” (Mitsubishi ChemicalIndustries Limited, Japan), “Amberlite XAD” (Rohm and Haas IndustriesU.S.A.), activated charcoal, or by ion exchange chromatography at pH 5to 9. The active material can be recovered from the cell mass byextraction with a water miscible solvent such as methanol, acetone,acetonitrile, n-propanol, or iso-propanol or with a water immisciblesolvent such as petroleum ether, dichloromethane, chloroform, ethylacetate or butanol. One other option is to extract the whole broth witha solvent selected from petroleum ether, dichloromethane, chloroform,ethyl acetate, methanol, acetone, acetonitrile, n-propanol,iso-propanol, or butanol. Typically, the active material is extractedwith ethyl acetate from the whole broth. Concentration andlyophilization of the extracts gives the active crude material.

The compounds of Formula I(a) and Formula I(b) of the present inventioncan be recovered from the crude material by fractionation using any ofthe following techniques: normal phase chromatography (using alumina orsilica gel as stationary phase; eluents such as petroleum ether, ethylacetate, dichloromethane, acetone, chloroform, methanol, or combinationsthereof); reverse phase chromatography (using reverse phase silica gelsuch as dimethyloctadecylsilyl silica gel, (RP-18) or dimethyloctylsilylsilica gel (RP-8) as stationary phase; and eluents such as water,buffers [for example, phosphate, acetate, citrate (pH 2 to 8)], andorganic solvents (for example, methanol, acetonitrile, acetone,tetrahydrofuran, or combinations of these solvents); gel permeationchromatography (using resins such as Sephadex LH-20® (Pharmacia ChemicalIndustries, Sweden), TSKgel® Toyopearl HW (TosoHaas, Tosoh Corporation,Japan) in solvents such as methanol, chloroform, acetone, ethyl acetate,or their combinations, or Sephadex® G-10 and G-25 in water); or bycounter-current chromatography (using a biphasic eluent system made upof two or more solvents such as water, methanol, ethanol, iso-propanol,n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride,chloroform, ethyl acetate, petroleum ether, benzene, and toluene). Thesetechniques may be used repeatedly, alone or in combination. A typicalmethod is chromatography over normal phase using silica gel.

The compounds of Formula I(a) and Formula I(b) and stereoisomersthereof, can be converted into their pharmaceutically acceptable saltsand derivatives which are all contemplated by the present invention.

Salts of the compounds can be prepared by standard procedures known toone skilled in the art, for example, salts like hydrochloride andsulphate salts, can be prepared by treating the compounds of FormulaI(a) and Formula I(b) and isomers thereof, with a suitable acid, forexample hydrochloric acid, sulphuric acid.

The compounds of Formula I(a) and Formula I(b) have antibacterialactivity against a wide range of bacterial strains. The compounds ofFormula I(a) and Formula I(b) also have antimycobacterial activityagainst MDR Mycobacterium tuberculosis strains such as M. tuberculosisH37Rv; M. tuberculosis Clinical isolate—S (Streptomycin), H (Isoniazidor Isonicotinyl hydrazine), R (Rifampicin) and E (Ethambutol)—Resistant;and M. tuberculosis Clinical isolate—S, H, R and E sensitive.

One or more of the compounds of Formula I(a) and Formula I(b),stereoisomers and their pharmaceutically acceptable salts thereof, aloneor together, can be administered to animals, such as mammals, includinghumans, as pharmaceuticals and in the form of pharmaceuticalcompositions. One or more of the compounds of Formula I(a) and FormulaI(b), stereoisomers and their pharmaceutically acceptable salts thereof,alone or together, can be administered prophylatically to a patient whois at risk for being infected by a bacterial infection. The patient maybe someone who may be exposed to the bacteria in a medical setting suchas in a hospital or in other setting where the bacteria may be present.

Accordingly, the present invention also relates to the compounds ofFormula I(a) and Formula I(b), their stereoisomers and theirpharmaceutically acceptable salts for use as pharmaceuticals and to theuse of the compounds of Formula I(a) and Formula I(b), stereoisomers andtheir pharmaceutically acceptable salts for the manufacture ofmedicaments having antibacterial activity.

The present invention further relates to pharmaceutical compositions,which contain an effective amount of one or more of the compounds ofFormula I(a) and Formula I(b) and/or stereoisomers and/or one or morepharmaceutically acceptable salts and/or derivatives thereof, togetherwith at least one pharmaceutically acceptable excipient or carrieruseful for preventing or treating bacterial infections.

The effective amount of the compounds of Formula I(a) and Formula I(b),or its stereoisomers, or its pharmaceutically acceptable salts or itsderivatives as the active ingredient in the pharmaceutical preparationsnormally is from about 0.01 mg to 1000 mg.

The present invention also relates to method of treating or preventing abacterial infection comprising administering to a mammal in need thereofan effective amount of one or more of the compounds of Formula I(a) andFormula I(b) and/or stereoisomers and/or one or more pharmaceuticallyacceptable salts thereof.

The present invention also relates to a method for the manufacture of amedicament containing one or more of the compounds of Formula I(a) andFormula I(b) and/or stereoisomers and/or one or more pharmaceuticallyacceptable salts thereof, for the treatment or prevention of diseasescaused by bacterial infections.

The compounds of the present invention are particularly useful asanti-bacterial agents. The present invention accordingly relates to theuse of one or more of the compounds of Formula I(a) and Formula I(b)and/or stereoisomers and/or one or more pharmaceutically acceptablesalts and/or derivatives thereof, for the manufacture of a medicamentfor the prevention or treatment of diseases caused by bacterialinfections.

The bacterial infections for the treatment of which the compounds of thepresent invention are used may be caused by bacteria belonging toStaphylococcus, Streptococcus, Enterococcus, Bacillus or Mycobacteriumspecies. The bacteria belonging to Staphylococcus species can bemethicillin-resistant or vancomycin resistant. The bacteria belonging toEnterococci species can be vancomycin resistant. The bacteria belongingto Mycobacterium species can be multi drug-resistant.

The term “Staphylococcus species” refers to Gram-positive bacteria,which appear as grape-like clusters when viewed through a microscope andas large, round, golden-yellow colonies, often with β-hemolysis, whengrown on blood agar plates. Species of Staphylococus includeStaphylococcus aureus.

The term “Streptococcus species” refers to a genus of spherical,Gram-positive bacteria, and a member of the phylum Firmicutes.Streptococci are lactic acid bacteria. Streptococcus species includesbacteria such as S. hemolyticus, S. mitis, S. salivarius, S. pneumoniae.Streptococcus species are responsible for infectious diseases such asmeningitis, bacterial pneumonia, endocarditis, erysipelas andnecrotizing fasciitis (‘flesh-eating’ bacterial infections).

The term “Enterococcus species” refers to a genus of lactic acidbacteria of the phylum Firmicutes. They are Gram-positive cocci whichoften occur in pairs (Diplococci for example Diplococcus pneumoniae).Enterococci are facultative anaerobic organisms.

The term “Bacillus species” refers to a large number of diverse,rod-shaped Gram positive bacteria that are motile by peritrichousflagella and are aerobic such as B. anthracis, B. subtilis or anaerobicsuch as Clostridium spp. for example C. difficile. These Bacilli belongto division Firmicutes.

The term “Mycobacterium species” refers to Gram-positive, non-motile,pleomorphic rods related to the actinomyces. Tuberculosis in humans iscaused by Mycobacterium tuberculosis. MDR-TB (multi-drug resistanttuberculosis) describes strains of tuberculosis that are resistant to atleast the two first-line TB drugs, isoniazid and rifampicin.

The compounds of the present invention can be administered orally,nasally, topically, parenterally such as subcutaneously,intramuscularly, intravenously, or by other modes of administration.

Pharmaceutical compositions which contain one or more of the compoundsof Formula I(a) and Formula I(b) or a stereoisomer or a pharmaceuticallyacceptable salt or a derivative thereof, optionally with otherpharmaceutically acceptable excipient or carrier, can be prepared bymixing the active compounds with one or more pharmaceutically acceptableexcipients and/or carriers such as, wetting agents, solubilisers such assurfactants, vehicles, tonicity agents, fillers, colorants, maskingflavors, lubricants, disintegrants, diluents, binders, plasticizers,emulsifiers, ointment bases, emollients, thickening agents, polymers,lipids, oils, cosolvents, complexation agents, or buffer substances, andconverting the mixture into a suitable pharmaceutical form such as, forexample, tablets, coated tablets, capsules, granules, powders, creams,ointments, gels, syrup, emulsions, suspensions, or solutions suitablefor injection used for parenteral administration.

Examples of excipients and/or carriers that may be mentioned arecremophor, poloxamer, benzalkonium chloride, sodium lauryl sulfate,dextrose, glycerin, magnesium stearate, polyethylene glycol, starch,dextrin, lactose, cellulose, carboxymethylcellulose sodium, talc,agar-agar, mineral oil, animal oil, vegetable oil, organic and mineralwaxes, paraffin, gels, propylene glycol, benzyl alcohol,dimethylacetamide, ethanol, polyglycols, tween 80, solutol HS 15, waterand saline. It is also possible to administer the active substances assuch, without vehicles or diluents, in a suitable form, for example, incapsules.

As is customary, the galenic formulation and the method ofadministration as well as the dosage range which are suitable in aspecific case depend on the species to be treated and on the state ofthe respective condition or disease, and can be optimized using methodsknown in the art. On average, the daily dose of active compound in apatient is 0.0005 mg to 50 mg per kg, typically 0.001 mg to 20 mg perkg.

The following are provided as illustrative examples of the presentinvention and do not limit the scope thereof.

Example 1 Isolation of Culture No. PM0626271 from Soil Collected fromAntarctic Region

a) Composition of the isolation medium:

Modified artificial sea water agar: Peptone 1.5 g, yeast extract 0.5 g,ferric chloride 0.007 g, 1.0 L water (750 mL artificial sea water+250 mLdemineralised water), agar powder 15.0 g, final pH (at 25° C.) 7.4 to7.8.

Composition of the artificial seawater: Sodium chloride 24.6 g,potassium chloride 0.67 g, calcium chloride.2H₂O 1.36 g, magnesiumsulphate.7H₂O 6.29 g, magnesium chloride.6H₂O 4.66 g, sodium bicarbonate0.18 g, demineralised water 1.0 L, final pH (at 25° C.) 7.8 to 8.2.

b) Procedure:

From Schirmacher Oasis region in Antarctica area, surface level soil wascollected and was stored at −20° C. throughout the journey to PiramalLife Sciences Limited, Goregaon, Mumbai, India. The sample was stored at−20° C. to −22° C. and later thawed to room temperature (25±2° C.) forisolation of the microbes. The soil sample (˜1 g) was suspended in 25 mLof sterile 1% peptone water in a 100 mL sterilized flask. The flask wasvortexed for 30 seconds. Serial dilutions up to 10⁻⁵ were prepared insterile 1% peptone water. 100 μL of 10⁻⁵ dilution was surface spread onmodified artificial seawater agar. The plate was incubated at roomtemperature (25±2° C.) till colonies were observed. After incubation forone and a half month, the colony which appeared on this medium wasstreaked on petri plates containing actinomycete isolation agar [HiMedia] prepared in 75% artificial sea water [Accumix™](AS-AIA). Theisolate was purified and was provided culture ID number PM0626271. Theculture no. PM0626271 was thus isolated from amongst the growingmicroorganisms as single isolate.

Example 2 Purification of Culture No. PM0626271

a) Composition of the purification medium (Actinomycete Isolation Agar,agarified by 1.5% agar agar):

Glycerol 5.0 mL, sodium caseinate 2.0 g, L-asparagine 0.1 g, sodiumpropionate 4.0 g, dipotassium phosphate 0.5 g, magnesium sulphate 0.1 g,ferrous sulphate 0.001 g, 1.0 L water (750 mL Artificial Sea Water+250mL demineralised water), agar powder 15.0 g, final pH (at 25° C.) 7.4 to7.8.

Composition of the artificial seawater: Sodium chloride 24.6 g,potassium chloride 0.67 g, calcium chloride.2H₂O, 1.36 g, magnesiumsulphate.7H₂O 6.29 g, magnesium chloride.6H₂O 4.66 g, sodium bicarbonate0.18 g, demineralized water 1.0 L, final pH (at 25° C.) 7.8 to 8.2.

b) Procedure:

The culture no. PM0626271 was streaked on Actinomycete Isolation Agar(containing 75% artificial sea water salts) petriplate. The petriplatewas incubated for 10 days at 25° C. One of the isolated colonies fromthe petriplate was transferred to fresh slants of Actinomycete IsolationAgar prepared in 75% artificial seawater. The slants were incubated for10 days at 25° C.

Example 3 Maintenance of Producer Strain—Culture No. PM0626271

a) Composition of the medium (Actinomycete Isolation Agar):

Glycerol 5.0 mL, sodium caseinate 2.0 g, L-asparagine 0.1 g, sodiumpropionate 4.0 g, dipotassium phosphate 0.5 g, magnesium sulphate 0.1 g,ferrous sulphate 0.001 g, 1.0 L water (750 mL artificial sea water+250mL demineralised water), agar powder 15.0 g, final pH (at 25° C.) 7.4 to7.8.

Composition of the artificial sea water: Sodium chloride 24.6 g,potassium chloride 0.67 g, calcium chloride.2H₂O 1.36 g, magnesiumsulphate.7H₂O 6.29 g, magnesium chloride.6H₂O 4.66 g, sodium bicarbonate0.18 g, demineralized water 1.0 L, final pH (at 25° C.) 7.8 to 8.2.

b) After dissolving the ingredients thoroughly by heating, the resultantsolution was distributed in test tubes and sterilized at 121° C. for 30minutes. The test tubes were cooled and allowed to solidify in aslanting position. The agar slants were streaked with the growth ofculture no. PM0626271 by a wire loop and incubated at 27° C. to 29° C.until a good growth was observed. The well-grown cultures were stored inthe refrigerator at 4° C. to 8° C.

Example 4 Fermentation of the Culture No. PM0626271 in Shake Flasks

a) Composition of seed medium [AS-274 (1)]:

Glucose 15 g, corn steep liquor 5 g, peptone 7.5 g, yeast extract 7.5 g,calcium carbonate 2.0 g, sodium chloride 5.0 g, volume made with 750 mLartificial sea water and 250 mL demineralised water.

b) The above medium was distributed in 40 mL amounts in 500 mL capacityErlenmeyer flasks and autoclaved at 121° C. for 30 minutes. The flaskswere cooled to room temperature (25±2° C.) and each flask was inoculatedwith a loopful of the well-grown producing strain (culture no.PM0626271) on the slant and shaken on a rotary shaker for 72 hours at230 rpm to 250 rpm at 30° C. (±1° C.) to give seed culture.

c) Composition of the production medium [AS 36P (1)]:

Soluble Starch 20 g, glucose 15 g, yeast extract 2 g, peptone 3 g,calcium carbonate 2 g, ammonium sulfate 0.5 g, corn steep liquor 2 g,sodium chloride 2 g, magnesium phosphate 5 g, cobalt chloride 1 mL/Lfrom stock of 1 g/L, trace salt solution 1 mL/L, volume made to 1 Lusing with 75% artificial sea water and 25% demineralised water.

d) 40 mL of the production media in 500 mL capacity Erlenmeyer flaskswas autoclaved at 121° C. for 30 minutes, cooled to 29° C. to 30° C. andseeded with 5% (v/v) of the seed culture mentioned in Example 4b.e) Fermentation parameters:

The production flasks were incubated on shaker at 29° C. and 220 rpm for96 hours. The production flasks were harvested and the whole broth fromeach media flask was extracted with equal volume of methanol undershaking condition for one hour at 29° C. and centrifuged at 3500 rpm forhalf an hour. The supernatant was used for antibacterial agar welldiffusion assay for monitoring of the activity.

The production of the compounds of Formula I(a) and Formula I(b) in thefermentation broth was determined by testing the bioactivity against S.aureus E710 (MRSA strain) and/or Enterococcus faecium R2 (VRE) using theagar well diffusion method. The harvest pH of the fermented broth was7.0 to 8.0. The fermented broth was harvested and the whole broth wasused for isolation and purification of the compounds of Formula I(a) andFormula I(b).

Example 5 Preparation of Seed Culture in Shake Flasks for Fermentation

a) Composition of the medium [AS-274 (1)]:

Glucose 15 g, corn steep liquor 5 g, peptone 7.5 g, yeast extract 7.5 g,calcium carbonate 2.0 g, sodium chloride 5.0 g, volume made with 750 mLArtificial Sea Water and 250 mL demineralised water.

b) The above medium was distributed in 200 mL amounts in 1000 mLErlenmeyer flasks and autoclaved at 121° C. for 30 minutes. The flaskswere cooled to room temperature (25±2° C.) and each flask was inoculatedwith a loopful of the well-grown producing strain (PM0626271) on theslant and shaken on a rotary shaker for 70 hours to 74 hours at 230 rpmto 250 rpm at 29° C. to 30° C. to obtain the seed culture.

Example 6 Cultivation of the Culture No PM0626271 in Fermenter

a) Composition of the production medium:

Artificial Sea Water (artificial sea water salt 28.32 g) (75%), solublestarch 20 g, glucose 15 g, yeast extract 2 g, peptone 3 g, calciumcarbonate 2 g, ammonium sulphate 0.05 g, corn steep liquor 2 g, sodiumchloride 2 g, magnesium phosphate 5 g, cobalt chloride (cobalt chloride1 g demineralized water 1.0 L) 1 mL/L, trace salt solution (coppersulphate 7 g, ferrous sulphate 1 g, manganese chloride 8 g, zincsulphate 2 g, demineralized water 1.0 L) 1 mL/L, demineralized water 1.0L, pH 6.5 to 7.5 (before sterilization).

b) 100 L of the production medium in 150 L fermenter along with 30 mL ofdesmophen as an antifoaming agent was sterilized in situ for 30 minutesat 121° C., cooled to 29° C. to 30° C. and seeded with 2.5 L to 3.5 L ofthe seed culture obtained above (Example 5).c) Fermentation parameters: The fermentation was carried out attemperature 29° C. to 30° C., agitation 100 rpm, aeration 60 lpm andharvested at 70 hours to 74 hours. The production of the compounds ofFormula I(a) and Formula I(b) in the fermentation broth was detectedqualitatively by testing the bioactivity against S. aureus E710 (MRSAstrain) and/or Enterococcus faecium R2 (VRE) using the agar welldiffusion method. The harvest pH of the fermented broth was 7.5 to 8.0.After the harvest, whole broth was subjected to solvent extraction.

Example 7 Isolation and Purification of the Compounds of Formula I(a)and Formula I(b)

The whole broth (10 L batch) was extracted using ethyl acetate (1:1).The organic and aqueous layers were separated. The organic layer wasprocessed to evaporate the solvent to obtain crude ethyl acetate extract(1.5 g). The crude extract was further processed by flash chromatography(silica gel, 30 g, solvent: methanol/chloroform step gradient, flow: 15mL/minute). The active compound eluted with 1% methanol to 5% methanolin chloroform, which was concentrated to obtain the semipure compound(250 mg). Further purification was carried out by repeated normal phasepreparative HPLC.

Preparative HPLC conditions:

-   -   Column: Eurospher silica (10μ, 20×250 mm)    -   Eluent: methanol:chloroform (5:95)    -   Flow rate: 20 mL/minute    -   Detection (UV): 245 nm    -   Retention time: compound of Formula I(a) (5 to 6 minutes),        -   compound of Formula I(b) (8 to 10 minutes)

Purity of fractions was checked by bioassay against E. faecium R2 and/orS. aureus 3066 and/or analytical HPLC. The eluates containing thecompounds of Formula I(a) and Formula I(b) were pooled and concentratedunder reduced pressure to remove the solvent to obtain compound ofFormula I(a) (40 mg), and compound of Formula I(b) (3 mg).

Analytical HPLC conditions:

-   -   Column: Eurospher RP-18, (3μ, 4.6×125 mm)    -   Solvent system: Gradient (0% acetonitrile to 100% in 15 minutes        against water, followed by 100% acetonitrile for 5 minutes)    -   Flow rate: 1 mL/minute    -   Detection (UV): 245 nm    -   Retention time: compound of Formula I(a) (12 to 13 minutes), and        compound of Formula I(b) (11 to 12 minutes)

A. Physical & Spectral Properties of the Compound of Formula I(a):

-   -   Appearance: White powder    -   Melting point: 240° C. (decomposes)    -   Solubility: Soluble in chloroform, ethyl acetate, methanol and        insoluble in water    -   MS [HR-ESI(+) MS)]m/z: 1650.4858 (M+H)    -   Molecular weight: 1649.5    -   Molecular formula: C₇₁H₆₃N₁₉O₁₈S₅    -   IR (KBr): 3386, 2927, 1648, 1507, 1206, 756, 666 cm⁻¹    -   ¹H NMR: refer to Table 1 and FIG. 1    -   ¹³C NMR: refer to Table 2 and FIG. 2

B. Physical & Spectral Properties of the Compound of Formula I(b):

-   -   Appearance: White powder    -   Solubility: Soluble in chloroform, ethyl acetate, methanol and        insoluble in water    -   MS [HR-ESI(+) MS)]m/z: 1674.4787 (M+Na)    -   Molecular weight: 1651.50    -   Molecular formula: C₇₁H₈₅N₁₉O₁₈S₅    -   ¹H NMR: refer to Table 3 and FIG. 3

TABLE 1 ¹H NMR of the compound of Formula I(a) in CDCl₃:CD₃OD (4:1) at500 MHz Peak δ 1 0.7 (d, 3H) 2 0.74 (d, 3H) 3 0.95 (d, 3H) 4 1.04 (s,3H) 5 1.08 (d, 3H) 6 1.2 (d, 3H) 7 1.28 (d, 3H) 8 1.34 (d, 3H) 9 1.37(m, 1H) 10 1.5 (d, 3H) 11 1.6 (d, 3H) 12 2.1 (m, 1H) 13 2.2 (m, 1H) 142.2 (m, 1H) 3.99 (m, 1H) 15 2.8 (d, 1H) 16 3.05 (t, 1H) 3.5 (t, 1H) 173.49 (d, 2H) 18 3.67 (d, 1H) 19 3.7 (q, 1H) 20 4.33 (d, 1H) 21 4.33 (d,1H) 22 4.62 ((q, 1H) 23 4.86 (dd, 1H) 24 5.19 (s, 1H) 25 5.19 (s, 1H),5.67 (s, 1H) 26 5.2 (t, 1H) 27 5.6 (d, 1H) 28 5.62 (s, 1H), 6.44 (s, 1H)29 5.65 (d, 2H) 30 5.72 (s, 1H), 6.61 (s, 1H) 31 6.1 (q, 1H) 32 6.25 (m,1H) 33 6.28 (d, 2H) 34 6.8 (d, 1H) 35 6.91 (s, 1H) 36 6.94 (s, 1H) 377.2 (s, 1H) 38 7.43 (s, 1H) 39 7.45 (s, 1H) 40 7.65 (s, 1H) 41 7.87 (s,1H) 42 8.05 (s, 1H) 43 8.17 (s, 1H) 44 8.2 (s, 1H) 45 8.5 (s, 1H) 468.67 (s, 1H) 47 8.99 (s, 2H) 48 9.72 (s, 1H) 49 9.8 (s, 1H)

TABLE 2 ¹³C NMR of the compound of Formula I(a) in CDCl₃:CD₃OD (4:1) at75 MHz Signal δ 1 12.11 2 13.74 3 14.1 4 14.8 5 16.48 6 17.11 7 17.27 817.55 9 20.9 10 23.13 11 27.56 12 27.56 13 29.04 14 33.24 15 46.17 1650.14 17 51.3 18 53.91 19 53.91 20 55.8 21 57.32 22 58.8 23 62.42 2462.73 25 64.45 26 64.73 27 65.69 28 65.95 29 70.27 30 77.1 31 101.45 32101.45 33 102.6 34 116.52 35 120.63 36 121.59 37 123.28 38 123.87 39123.87 40 125.48 41 126.03 42 126.69 43 128.24 44 130.34 45 130.98 46131.14 47 132.39 48 141.85 49 144.61 50 148.17 51 148.45 52 151.89 53152.76 54 155.45 55 157.9 56 159.0 57 160.04 58 160.36 59 161.01 60163.75 61 164.46 62 164.5 63 166.6 64 167.13 65 167.95 66 168.47 67168.67 68 170.35 69 170.35 70 171.63 71 172.0

TABLE 3 ¹H NMR of the compound of Formula I(b) in CDCl₃:CD₃OD (4:1) at500 MHz Peak δ 1 0.88 (d, 3H) 2 0.95 (d, 3H) 3 0.99 (d, 3H) 4 1.0 (m,1H) 5 1.16 (s, 3H) 6 1.18 (d, 3H) 7 1.32 (d, 3H) 8 1.35 (d, 3H) 9 1.43(d, 3H) 10 1.5 (d, 3H) 11 1.61 (d, 3H) 12 1.72 (d, 3H) 13 2.02 (m, 1H)14 2.28 (m, 1H) 15 2.8 (d, 1H) 16 3.17 (t, 1H) 3.69 (t, 1H) 17 3.43 (d,1H) 18 3.46 (d, 1H) 19 3.58 (d, 1H) 20 3.8 (q, 2H) 21 4.06 (m, 1H) 224.45 ((d, 1H) 23 4.59 (t, 1H) 24 4.61 (dd, 1H) 25 4.74 (t, 1H) 26 4.95(t, 1H) 27 5.19 (s, 1H), 5.78 (s 1H) 28 5.19 (s, 1H) 29 5.3 (d, 1H) 305.52 (s, 1H), 6.65 (s, 1H) 31 5.65 (s, 1H) 32 5.72 (d, 1H) 33 5.8 (d,1H) 34 6.1 (q, 1H) 35 6.34 (m, 1H) 36 6.36 (d, 1H) 37 6.73 (d, 1H) 386.91 (d, 1H) 39 6,94 (s, 1H) 40 7.14 (s, 1H) 41 7.3 (s, 1H) 42 7.46 (s,1H) 43 7.54 (d 1H) 44 7.65 (d, 1H) 45 7.80 (s, 1H) 46 8.05 (s, 1H) 478.11 (s, 1H) 48 8.14 (s, 1H) 49 8.26 (s, 1H) 50 8.52 (t, 1H) 51 8.65 (d,1H) 52 9.2 (s, 1H) 53 9.87 (s, 1H) 54 9.92 (s, 1H)

Biological Evaluation of the Compounds of Formula I(a) and Formula I(b)In-Vitro Assays Example 8

The in-vitro potency was established by minimum inhibitory concentration(MIC) determinations of the compounds of Formula I(a) and Formula I(b)against bacterial strains, by using the Macro-broth dilution method asper National Committee for Clinical Laboratory Standards (2000)guidelines (Methods for Dilution Antimicrobial Susceptibility Tests forBacteria that Grow Aerobically—Fifth Edition Approved Standard M7-A5.NCCLS, Wayne, Pa., USA). Mueller-Hinton broth was used as nutrientmedium for the assay, unless stated otherwise. PM181104 (PCT publicationno. WO2007119201) was used as known standard in all in-vitroexperiments. For preparation of the stock solution the compounds ofFormula I(a) and Formula I(b) were dissolved in chloroform (5% of thetotal required volume) and diluted using methanol (95% of the totalrequired volume).

Result:

The results obtained are shown in Table 4 and Table 5, and demonstratethat the compounds of Formula I(a) and Formula I(b) have utility intreating bacterial infections.

TABLE 4 MICS of the compound of Formula I(a) against bacterial strainsMIC Test Organism (μg/ml) Test Organism MIC (μg/ml) S. aureus C1 MRSA 20.25 S. aureus E712, MRSA 0.25 Erythro^(R), 59 S. aureus C1 MRSA 3 0.25S. aureus 503, MRSA, 62 >1 S. aureus C1 MRSA 5 0.25 S. aureus SG 511,MRSA, 63 1 S. aureus C1 MRSA 7 0.125 S. aureus 789, MRSA, 64 >1 S.aureus C1 MRSA 8 0.25 S. aureus 209 P, MSSA 0.063 S. aureus C1 MRSA 90.25 S. epidermidis 823, Teicho^(R), 230 0.25 S. aureus C1 MRSA 10 0.25S. epidermidis 6098, Erythro^(R), 233 >1 S. aureus C1 MRSA 13 0.25 S.epidermidis 6729 II W, 0.125 Erythro^(R), 236 S. aureus C1 MRSA 16 0.25S. epidermidis 2361 W, 246 0.25 S. aureus C1 MRSA 17 0.125 S.epidermidis 4264 I (1) W, 0.125 247 S. aureus C1 MRSA 20 0.125 S.epidermidis Pat 01 IV, 251 0.25 S. aureus C1 MRSA 21 0.125 E. faecium C1VRE 26 0.25 S. aureus C1 MRSA 22 0.25 E. faecium C1 VRE 27 0.25 S.aureus C1 MRSA 23 0.016 E. faecium C1 VRE 28 0.125 S. aureus C1 MRSA 240.25 E. faecium C1 VRE 31 0.5 S. aureus C1 MRSA 25 0.15 E. faecium C1VRE 33 0.125 S. aureus KEM MRSA 1 0.25 E. faecium C1 VRE 34 0.25 S.aureus KEM MRSA 2 0.25 E. faecium KEM VRE 1 0.25 S. aureus KEM MRSA 30.25 E. faecium KEM VRE 2 0.5 S. aureus KEM MRSA 4 0.125 E. faecium KEMVRE 3 0.25 S. aureus KEM MRSA 5 0.125 E. faecium KEM VRE 4 0.5 S. aureusMRSA 3 lilavati 0.125 E. faecium KEM VRE 5 0.25 S. aureus Misk MRSA 350.125 E. faecium R-2 (VRE), 0.25 S. aureus Misk MRSA 37 0.125Enterococcus faecium, VSE 0.125 (322) S. aureus Misk MRSA 38 0.125Bacillus cereus (121) 0.031 S. aureus E710, MRSA 0.125 Bacillus subtilisATCC 6633 0.031 (123) S. aureus ATCC 33591, 0.125 Bacillus megateriumFH1127 0.125 MRSA (124)

TABLE 5 MICs of the compounds of Formula I(a) and Formula I(b) againstbacterial strains Minimal Inhibitory Concentration (μg/ml) Compound ofCompound of Test culture Formula I(a) Formula I(b) E. faecium, R-2 (VRE)0.125 2 S. aureus E710, (MRSA) 0.125 2 S. aureus 209P (MSSA) 0.064-0.1250.25-0.5

Abbreviations used in Table 4 and Table 5 are—

S: Staphylococcus E: Enterococci Example 9 Evaluation ofAntimycobacterial Activity

The assay was done as reported in J. Clin. Microbiol., 1999, 37, 1144.

50 μL of bacterial (as mentioned in Table 6) suspension, equivalent toMacFarlands no. 2 standard (corresponding to >5×10⁷ CFU/ml) (Remel,Lenexa, Kan.) was added to 400 μL of G7H9 with and without compounds ofFormula I(a) and Formula I(b) (tested at 0.5 μg/mL, 5 μg/mL and 10μg/mL) and incubated for 72 hours at 37° C. After incubation 50 μL ofthe high titer Luciferase reporter phage (phAE 129) and 40 μL of 0.1Mcalcium chloride (CaCl₂) were added to all the vials and this set up wasincubated for 4 hours at 37° C. After incubation 100 μL of the mixturewas taken from each vial into a luminometer cuvette and equal amount ofworking D-luciferin (0.3 mM in 0.05 M sodium citrate buffer, pH 4.5)solution was added. The Relative Light Units (RLUs) were measured after10 seconds of integration in the Luminometer (Monolight 2010).

Duplicate readings were recorded for each sample and the mean wascalculated. The percentage reduction in the RLU was calculated for eachtest sample and compared with control. The experiment was repeated whenthe mean RLU of the control was less than 1000. The criterion foractivity is antimycobacterial activity indicated by fifty percentreduction in RLU in the presence of the compound in comparison withcompound free control.

TABLE 6 Antimycobacterial activity of the compounds of Formula I(a) andFormula I(b) % reduction in RLU Strain Compounds 0.5 μg/mL 5 μg/mL 10μg/mL M. tuberculosis Compound of 36.36 77.17 83.76 H₃₇Rv Formula I(a)Compound of 28.87 54.83 76.73 Formula I(b) Clinical isolate: Compound of20.88 78.11 88.42 S, H, R and E Formula I(a) resistant Compound of 5.6770.9 83.74 Formula I(b)

CONCLUSION

The compounds of Formula I(a) and Formula I(b) are active againststandard strain of TB (H₃₇ RV) and MDR Mycobacterium tuberculosisstrains [resistant to 4 standard antibiotics: S (Streptomycin), H(Isoniazid or Isonicotinyl hydrazine), R (Rifampicin) and E(Ethambutol)].

1-19. (canceled)
 20. An isolated compound selected from the compound ofFormula I(a) or the compound of Formula I(b):

or a stereoisomer, or a tautomer, or a pharmaceutically acceptable saltthereof.
 21. The isolated compound as claimed in claim 20, wherein thecompound is isolated from fermented broth of a microorganism belongingto Streptomyces species (PM0626271/MTCC 5447).
 22. The isolated compoundof Formula I(a) as claimed in claim 20, wherein said compound ischaracterized by: (a) molecular weight of 1649.5, (b) molecular formulaC₇₁H₈₃N₁₉O₁₈S₅, (c) ¹H NMR spectrum as depicted in FIG. 1, and (d) ¹³CNMR spectrum as depicted in FIG.
 2. 23. The isolated compound of FormulaI(b) as claimed in claim 20, wherein said compound is characterized by:(a) molecular weight of 1651.5, (b) molecular formula C₇₁H₈₅N₁₉O₁₈S₅,and (c) ¹H NMR spectrum as depicted in FIG.
 3. 24. A pharmaceuticalcomposition comprising an effective amount of the isolated compound ofFormula I(a) or the isolated compound of Formula I(b) as claimed inclaim 20 and at least one pharmaceutically acceptable excipient orcarrier.
 25. The pharmaceutical composition as claimed in claim 24,wherein the pharmaceutical composition is in the form of a tablet,coated tablet, capsule, granule, powder cream, ointment, gel, emulsion,suspension, or solution for injection.
 26. A method of treating orpreventing a bacterial infection comprising administering to a mammal inneed thereof an effective amount of the isolated compound of FormulaI(a) or the isolated compound of Formula I(b) as claimed in claim 20.27. The method as claimed in claim 26, wherein the bacterial infectionis caused by bacteria belonging to Staphylococcus, Streptococcus,Enterococci, Bacillus or Mycobacterium species.
 28. The method asclaimed in claim 27, wherein the bacteria belonging to Staphylococcusspecies is methicillin-resistant, or vancomycin-resistant or both. 29.The method as claimed in claim 27, wherein the bacteria belonging toEnterococci species is vancomycin-resistant.
 30. The method as claimedin claim 27, wherein the bacteria belonging to Mycobacterium species ismulti drug-resistant.